Tamsulosin, an α1-adrenoceptor antagonist, and sildenafil, a phosphodiesterase (PDE) inhibitor, are reported to improve lower urinary tract symptoms including overactive bladder (OAB). This study is aimed at investing the effects of tamsulosin and sildenafil and comparing the degree of the suppressive effects on the afferent pathways of micturition between them using an animal model of OAB, the spontaneously hypertensive rat (SHR).The cystometric parameters, the basal pressure and duration of bladder contraction, were significantly increased in the SHR group as compared with the Wistar-Kyoto (WKY) group. The intercontraction interval also significantly decreased in the SHR group. In the SHR-Tam 0.01 mg/kg group and the SHR-Sil 1 mg/kg group, however, the basal pressure and duration were significantly reduced and the intercontraction interval was significantly prolonged. Moreover, the degree of the expression of c-Fos and NGF was significantly higher in the SHR group as compared with the WKY group. But it was significantly reduced in the SHR-Tam 0.01 mg/kg group and the SHR-Sil 1 mg/kg group. Furthermore, tamsulosin had a higher degree of effect as compared with sildenafil.In conclusion, α1-adrenergic receptor antagonists and PDE-5 inhibitors may have an effect in improving the voiding functions through an inhibition of the neuronal activity in the afferent pathways of micturition. Overactive bladder (OAB) syndrome is clinically diagnosed based on the presence of urinary urgency. In addiThe bladder and external urethral sphincter are innervated directly or indirectly by the nerve fibers arising from several brain regions including the pontine micturition center (PMC), hypothalamus and preoptic area. The PMCc-Fos is an immediate early gene, and its expression is triggered by stimuli-induced changes in the metabolic activity of neurons under various conditions. It servOne of the α1-adrenoceptor blockers, tamsulosin is considered the most effective regimen for patients with lower urinary tract symptoms (LUTS) that are suggestive of benign prostatic hyperplasia. Long-teAlthough many studies have reported the efficacies of α1-adrenergic receptor blockers and PDE inhibitors on storage and voiding symptoms, their exact mechanisms of action based on the expression of neuronal activity markers remain unclear-13. MoreIn this study, we investigated the effects of tamsulosin and sildenafil and compared the degree of the suppressive effects on the afferent pathways of micturition  and PMC) between them.We used a total of 32 rats, aged 10 weeks, weighing 250±10 g  in accordance with the animal care guidelines of the National Institutes of Health (NIH) and the Korean Academy of Medical Sciences (KAMS). We obtained IRB  approval for our study (IRB# 1103–05). We randomly divided our experimental animals into the following four groups:(1) The WKY group (the control group) (n=8): Water (1 mL) as the vehicle.(2) The SHR group (n=8): Water (1 mL) as the vehicle.(3) The SHR-Tam 0.01 mg/kg group (n=8): A 4-week daily course of oral tamsulosin at a dose of 0.01 mg/kg(4) The SHR-Sil 1 mg/kg group (n=8): A 4-week daily course of oral sildenafil at a dose of 1 mg/kg.tiletamine hydrochloride and zolazepam hydrochloride. The rats underwent implantation of a sterile polyethylene catheter (PE50) into the femoral artery, connected to a pressure transducer . Blood pressure monitoring was done using Labscribe .Both the SHR and WKY rats were anesthetized with an intraperitoneal injection of Zoletil 50® (10 mg/kg)  composed of 2O were analyzed.We performed cystometry as previously described. Here, tFollowing the cystometry, the rats were transcardially perfused with 50 mM phosphate-buffered saline (PBS) and then fixed using 4% paraformaldehyde in 100 mM sodium phosphate buffer at pH 7.4. The brain was extracted, postfixed in the same fixative overnight and transferred to a 30% sucrose solution for cryoprotection. This was followed by the preparation of serial coronal sections of 40 μm in thickness using a freezing microtome. The vlPAG and the PMC were selected from the midbrain region spanning from Bregma -7.64 to -8.00 mm and from Bregma -9.68 to -9.80 mm, respectively. Then, the dorsal horn of the L5 spinal cord was selected. In each region, four sections were collected on average from each rat.2O2 in 50 mM Tris–HCl (pH 7.6). Then, the tissue samples were rinsed with PBS three times and mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and the coverslips were mounted by using Permount® .To analyze the degree of the expression of c-Fos and NGF in the afferent pathways of micturition, we performed immunohistochemistry as previously described. Coronal2 in each region.The degree of the expression of c-Fos and NGF in the afferent pathways of micturition was analyzed based on the number of c-Fos- or NGF-positive cells using the Image-Pro® Plus computer-assisted image analysis system  installed in a light microscope . We expressed the results as the number of cells/mmpost-hoc analysis. A P-value of <0.05 was considered statistically significant.Statistical analysis was performed using IBM SPSS . All data are expressed as the mean±SEM (SEM: standard error of the mean). We also performed the one-way ANOVA and Duncan’s P<0.05). In addition, the basal pressure and duration of bladder contraction were significantly higher and longer in the SHR group (P<0.05). But they were significantly reduced in the SHR-Tam 0.01 mg/kg group and the SHR-Sil 1 mg/kg (P<0.05). These results indicate that the basal pressure and duration of bladder contraction were significantly reduced following the treatment with tamsulosin or sildenafil. Moreover, Tam 0.01 mg/kg and Sil 1 mg/kg significantly prolonged the ICI (P<0.05)  significantly decreased in the SHR group as compared with WKY group (P<0.05). But it was significantly reduced in the SHR-Tam 0.01 mg/kg group and the SHR-Sil 1 mg/kg group (P<0.05) . On the other hand, it was significantly reduced in the SHR-Tam 0.01 mg/kg group and the SHR-Sil 1 mg/kg group (P<0.05) . But it was significantly reduced in the SHR-Tam 0.01 mg/kg group and the SHR-Sil 1 mg/kg group (P<0.05)  and the increased occurrence of non-voiding contractions that are suggestive of detrusor overactivity. Presumavia the spinal reflex pathway[via the hypogastric, pelvic and pudendal nerves[via the neurons in the lumbosacral region of the spinal cord when the bladder is filled with urine. This is followed by the activation of the cells sending a projection to the PMC of the PAG, followed by the micturition[According to de Groat and Yoshimura, the expression of c-Fos in the spinal cord is an indicator of the involvement of the spinal neurons in processing afferent signals from the lower urinary tract  pathway. Afferenl nerves. It can l nerves. Then, tturition,18. In cturition. The ponturition,20. Withvia the micturition reflex pathway, and it plays a vital role in the pathogenesis of bladder overactivity at the spinal level[+ current density as well as elevated NGF levels in the bladder afferent neurons[NGF modulates the neuronal function al level. Its leval level,22. The  neurons. Our res neurons.With the activation of the α1-adrenergic receptor in the bladder, the OAB symptoms are presented. It can therefore be inferred that α1-adrenergic receptor antagonists might be effective in improving the micturition functions. It has It is well known that PDE-5 inhibitors improve the LUTS, whose effects might be maximized during bladder filling,29,30. SThere is a possibility that α1-adrenergic receptor antagonists and PDE inhibitors may improve the LUTS including OAB. Still, Based on our results, it can be concluded that α1-adrenergic receptor antagonists and PDE-5 inhibitors may have an effect in improving the voiding functions through an inhibition of the neuronal activity in the dorsal horn of the L5 spinal cord, vlPAG and PMC.The limitations of the current study are as follows:(1) We demonstrated that α1-adrenergic receptor antagonist (tamsulosin) and PDE-5 inhibitor (sildenafil) acted on the afferent pathways of micturition and thereby suppressed their effects on the micturition reflex. But we failed to clarify the exact mechanisms that are involved in other micturition regions such as the pelvis and pudendal afferent and efferent nerves, the anterior cingulate gyrus (ACG), medial preoptic nucleus (MPA) and frontal cortex.(2) We compared the suppressive effects on the neuronal activity between the two drugs in a monotherapy setting. Further animal experimental studies are therefore warranted to compare the suppressive effects on the neuronal activity between α1-adrenergic receptor antagonists and PDE-5 inhibitors when concomitantly used.(3) We failed to evaluate the dose-effect relationship of sildenafil, which deserves further studies. To clarify the dose-effect relationship, both drugs should be treated at optimal dose. In our preliminary study, we administered tamsulosin and sildenafil at varying doses of 0.01 mg.kg/day, 0.1 mg/kg/day and 1 mg/kg/day and 1 mg/kg/day, 5 mg/kg/day and 10 mg/kg/day, respectively. Thus, we found that the degree of efficacy of tamsulosin and sildenafil was highest when administered at doses of 0.01 mg/kg/day and 1 mg/kg/day, respectively.In summary, we demonstrated that α1-adrenergic receptor antagonists and PDE-5 inhibitors may have an effect in improving the voiding functions through an inhibition of the neuronal activity in the afferent pathways of micturition, such as the dorsal horn of the L5 spinal cord, vlPAG and PMC. Moreover, tamsulosin was more effective in alleviating the OAB symptoms by suppressing more neuronal activity in the afferent pathways of micturition as compared with sildenafil. But further prospective clinical studies are warranted to establish their efficacy in patients with OAB.ACG: Anterior cingulate gyrus; cGMP: Cyclic guanosine monophosphate; DAB: 3,3′-diaminobenzidine; L5: 5th of lumbar vertebra; LUTS: Lower urinary tract symptoms; MCA: Middle cerebral artery; MPA: Medial preoptic nucleus; NGF: Nerve growth factor; NO: Nitric oxide; OAB: Overactive bladder; PAG: Periaqueductal gray matter; PBS: Phosphate-buffered saline; PDE: Phosphodiesterase; PDE-5: Phosphodiesterase-5; PMC: Pontine micturition center; SHR: Spontaneously hypertensive rat; Sil: Sildenafil; Tam: Tamsulosin; vlPAG: Ventrolateral periaquaductal gray; WKY: Wistar-kyoto.The authors declare that they have no competing interests.K-HK conceived and designed this study; S-EK drafted the manuscript and acquired data; C-JK supervised this study and provided the critical revision of the manuscript for important intellectual content; Il-GK and M-SS analyzed and interpreted data; LH and In-YC provided the administrative, technical, or material support. All authors read and approved the final manuscript.


Catalytic upgrading reduced the amount of oxygenates in the product vapor due to the cracking reaction of the catalysts. Levoglucosan, a polymeric oxygenate species, was completely decomposed without being detected. While the amount of heavy phenols was reduced by catalytic upgrading, the amount of light phenols was increased because of the catalytic cracking of heavy phenols into light phenols and aromatics. The amount of aromatics increased remarkably as a result of catalytic upgrading, which is attributed to the strong Brönsted acid sites and the shape selectivity of the Meso-MFI catalyst. The addition of Pt made the Meso-MFI catalyst even more active in deoxygenation and in the production of aromatics.Catalytic fast pyrolysis of waste rice husk was carried out using pyrolysis-gas chromatography/mass spectrometry [Py-GC/MS]. Meso-MFI zeolite [Meso-MFI] was used as the catalyst. In addition, a 0.5-wt.% platinum [Pt] was ion-exchanged into Meso-MFI to examine the effect of Pt addition. Using a catalytic upgrading method, the activities of the catalysts were evaluated in terms of product composition and deoxygenation. The structure and acid site characteristics of the catalysts were analyzed by Brunauer-Emmett-Teller surface area measurement and NH Due to the increasing cost of crude oil and the environmental problems stemming from the overuse of fossil fuels, it has become increasingly important to develop alternative forms of renewable energy. Among these, biomass is regarded as a promising renewable energy source, and research on its application is being conducted extensively all over the world. In principle, compared to conventional fossil fuels, biomass does not cause a net carbon dioxide increase in the atmosphere, and it contains lower amounts of sulfur and nitrogen. Therefore, the use of biomass has benefits in terms of mitigating climate change as well as addressing the problem of air pollution -3.2O, CO, and CO2 and removed to improve the bio-oil's quality  was used to analyze the pyrolysis products directly and to examine the effect of the catalyst.Rice husk was supplied from a rice mill located in Jeonnam, Korea. It was dried in an oven for 24 h at 110°C to minimize the effect of moisture. The sample particles were 8 to 10 mm long, 2.0 to 2.5 mm wide, and 0.1 to 0.15 mm thick. For each experiment, 1 mg of the sample was used.Ultimate analysis was performed using a TruSpec elemental analyzer  and an SC-432DR sulfur analyzer  to quantify C, H, O, N, and S. Proximate analysis was carried out using a thermogravimetric analyzer .3)4(NO3)2. Calcination for 3 h at 500°C under O2 atmosphere and H2 reduction was applied to the Pt-Meso-MFI before use  method.3 temperature-programmed desorption [TPD] analysis was carried out using a TPD/TPR 2900 analyzer . Before each analysis, the sample was pretreated with He gas at 250°C and was then cooled down to 100°C. While heating the sample from 100 to 400°C at a rate of 10°C/min with a N2 flow rate of 50 mL/min, the amount of NH3 desorbed from the catalyst was measured using a thermal conductivity detector.To investigate the acidic properties of the catalysts, NHPy-GC/MS experiments were performed using a vertical furnace type pyrolyzer . About 1 mg of the rice husk sample was put on the metal sample cup floor. An intermediate layer made of quartz wool was installed above the sample to prevent contact between the catalyst and the sample. About 1 mg of the catalyst was located above the quartz wool layer. Therefore, catalytic upgrading was supposed to take place when the vapor-phase pyrolysis products passed through the catalyst layer. The metal sample cup containing the catalyst and the sample was inserted into the preheated pyrolyzer through which helium carrier gas flowed.Vapor species produced from pyrolysis reactions for 3 min at 500°C were analyzed directly using a GC (HP 6890N Gas Chromatography)/MS  connected to the pyrolyzer. An HP-5 MS (30 m × 0.25 mm × 0.25 μm) capillary column was used for the analysis. Carrier gas was supplied with a split ratio of 50:1, and the GC/MS interface temperature was controlled at 300°C. The GC oven temperature was programmed to rise from 40 to 300°C at a rate of 5°C/min so that the analysis was conducted for 66 min, including 4 min maintained at 40°C before heating and 10 min maintained at 300°C after heating. Each peak appearing in the obtained mass spectra was interpreted using the NIST05 library.The physicochemical properties of waste rice husk are displayed in Table 2/g), and the pore size was about 4.1 nm, which is large enough to allow bio-oil molecules to diffuse into the pores. When Pt was added, the specific surface area decreased slightly due to the blocking of the pores by the Pt.Table 3-TPD] analysis performed to examine the acid properties of the catalysts. Meso-MFI exhibited two peaks: a low-temperature peak at about 230°C and a high-temperature peak at about 400°C. The low-temperature peak represents weak acid sites, while the high-temperature peak represents the strong Brönsted acid sites [Figure id sites . As withTo analyze the distribution of pyrolysis products easily and accurately, the Py-GC/MS method was used. Fast pyrolysis was carried out at 500°C, which has been suggested to be the optimal temperature for catalytic pyrolysis of biomass . It is wThe yields and compositions of the pyrolysis products were changed due to catalytic upgrading. Catalysts can affect the production of volatile liquid products via two pathways during the catalytic pyrolysis process. First, the product species can be cracked into gasses or converted into coke or char, which reduces the volatile liquid species. Second, catalysts decompose nonvolatile oligomers into volatile monomers, increasing the amount of volatile liquid species. The change in the liquid product yield due to catalytic upgrading is determined by a sum of these two opposite effects .1-C4 hydrocarbons, and H2O. In particular, when Pt was added, the reduction of the total peak area for the liquid product was larger due to a stronger cracking effect. This result indicates that catalytic upgrading reduces the liquid product yield.Total GC/MS peak areas of liquid products were plotted in Figure To investigate the change in the composition of the pyrolysis product due to catalytic upgrading, the pyrolysis products obtained with and without a catalyst were divided into seven groups, i.e., acids, hydrocarbons, oxygenates, phenolics, anhydrosugars, aromatics, and gas. Figure 2O by dehydration or into CO and CO2 by decarbonylation and decarboxylation. It was observed that catalytic upgrading resulted in an increased gas yield, levoglucosan was decomposed completely, and the amount of carbonyl-containing species was decreased (data not shown). The reduction of total oxygenates due to Meso-MFI and Pt-Meso-MFI was 38% and 49%, respectively. Oxygenates degrade the quality of bio-oil by reducing its heating value and producing air pollutants upon combustion. Therefore, a reduction of oxygenate production by catalytic upgrading may help to produce higher-quality bio-oil. In particular, Pt-Meso-MFI showed a higher deoxygenation effect, which is believed to be due to enhanced cracking, dehydrogenation, hydrogenolysis, and hydrocracking reactions over Pt [As shown in Figure  over Pt .The total amount of phenolics was reduced due to catalytic upgrading, but the amount of light phenols was increased. This result is attributed to cracking of heavy phenols into light phenols and aromatics due to the outstanding cracking ability of the Meso-MFI catalyst. Figure Aromatics that were not produced by non-catalytic pyrolysis appeared after catalytic upgrading. In particular, the amount of high-value-added mono-aromatics such as toluene and p-xylene increased remarkably Figure . The exiIn this study, catalytic pyrolysis of waste rice husk was carried out using Py/GC-MS. The catalytic activities of Meso-MFI and Pt-Meso-MFI were evaluated by comparing the product compositions with that of non-catalytic pyrolysis.The yield of oxygenates that can degrade the product bio-oil was reduced due to catalytic upgrading, by 38% with Meso-MFI and by 49% with Pt-Meso-MFI, demonstrating the outstanding activity of the Pt-Meso-MFI catalyst in deoxygenation. Catalytic upgrading over Meso-MFI increased the amount of light phenols, which is attributed to cracking of heavy phenols into light phenols and aromatics due to the catalytic effect of mesoporous Meso-MFI. In addition, Meso-MFI exhibited a good selectivity for aromatics, which is ascribed to its strong acid sites and shape selectivity due to the MFI structure. Pt-Meso-MFI was even more effective in the upgrading of bio-oil because of the additional catalytic effect of Pt.3-TPD: temperature programmed desorption of ammonia; Py-GC/MS: pyrolysis gas chromatography/mass spectrometry.Meso-MFI: meso-MFI zeolite; NHThe authors declare that they have no competing interests.MJJ, SSK, JKJ, SHP, JMK, JMS, and SHL participated in some of the studies and in drafting the manuscript. YKP conceived the study, participated in all experiments of this study, and prepared and approved the final manuscript.


Burkholderia kururiensis M130 is one of the few characterized rice endophytes and was isolated from surface-sterilized rice roots. This bacterium shows strong growth-promoting effects, being able to increase rice yields. Here we present its draft genome sequence, which contains important traits for endophytic life and plant growth promotion. Rice is the basic food for a large proportion of the world’s population . The proBurkholderia kururiensis M130, formerly named “Burkholderia brasilensis,” was isolated from surface-sterilized rice roots in Brazil; however, strains from the same species were also recovered from banana, pineapple, sugarcane, tobacco, and cassava  version 3.4.0 (B. kururiensis M130 presents a G+C content of 63% and, according to the automated annotation done using RAST (Genome sequencing was performed using a combination of Illumina  and 454 on 3.4.0  followeding RAST , containB. kururiensis M130 genome presents several genes related to plant growth promotion, including the accD gene encoding 1-aminocyclopropane-1-carboxylate deaminase, genes for the production of indole-3-acetic acid, and the nif gene cluster. The genome apparently does not possess genes for the production of antibiotics, and it presents one N-acylhomoserine lactone quorum-sensing system. The M130 genome shows a range of detoxification mechanisms, including for the degradation of organic substances, heavy metal efflux systems, and various enzymes necessary to cope with oxidative stress. In the present study, we determined the draft genome information of a strain of B. kururiensis with great biotechnological potential. To our knowledge, this is the first sequence report of this species.The ANSK00000000. The version described in this paper is the first version, ANSK01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


It is considered difficult to make a definitive diagnosis of focal nodular hyperplasia (FNH) of <3 cm when using conventional diagnostic imaging modalities. Typical FNH imaging findings are: i) central scar formation, ii) nutrient vessels extending radially from the center and iii) the presence of Kupffer cells. In a clinical setting, identification of a spoke-wheel pattern formed by nutrient vessels extending radially is a key feature in the diagnosis of FNH. In this study, we investigated the detection rate of spoke-wheel patterns of FNH <3 cm using arrival time parametric imaging (At-PI) technology with Sonazoid-enhanced ultrasonography (US). Five patients with FNH <3 cm who had undergone Sonazoid-enhanced US at the Toho University Omori Medical Center between February 2008 and March 2009 were included in the study. The mean tumor diameter was 20.2±7.2 mm. Lesions were enhanced with 0.5 ml Sonazoid US contrast agent and a video of the procedure was saved and used for At-PI analysis of contrast agent dynamics in FNH. Three ultrasonographic specialists examined the images and made a diagnosis of FNH based on the findings of spoke-wheel patterns. Similarly, micro-flow imaging (MFI) was performed to evaluate the contrast agent dynamics in FNH. Using MFI, FNH was diagnosed in 3 of the 5 cases by the three specialists, whereas At-PI enabled the identification of spoke-wheel patterns in all 5 cases. At-PI using Sonazoid-enhanced US is superior for detecting spoke-wheel patterns of FNH <3 cm. Focal nodular hyperplasia (FNH) is a benign hyperplastic lesion and the number of reports of FNH has been increasing owing to the widespread use of diagnostic imaging at routine medical check-ups. The characteristic imaging findings of FNH are: i) central scar formation, ii) nutrient vessels extending radially from the center and iii) the presence of Kupffer cells . When usIn this study, we used arrival time parametric imaging (At-PI), which enables color display of contrast agent dynamics in contrast-enhanced US to investigate the detection rate of spoke-wheel patterns in FNH <3 cm.Five patients  with FNH <3 cm who had undergone Sonazoid-contrast US at the Toho University Omori Medical Center in Tokyo, Japan between February 2008 and March 2009 were enrolled in the study. The mean tumor diameter was 20.2±7.2 mm . FNH wasUltrasonography was performed using a Toshiba Aplio XG ultrasound device  with a 3.75 MHz convex array probe (PVT-375 BT) at a mechanical index of 0.22–0.29. The focus was set at the bottom end of the tumors and 0.5 ml Sonazoid  was injected into the cubital vein. Data generated in the first 40 sec was saved as raw data on the hard disk. US was performed by the same operator to maintain imaging consistency.Following US, At-PI was performed using the proprietary image analysis software of the ultrasound system. The system set the point at which the contrast agent reached the hepatic tumor as time zero and sequentially calculated the arrival time at individual pixels representing the tumor. A color map was created and automatically superimposed on a B-mode image. Then, two parametric-color scales were used to analyze color patterns and the time setting for color distribution. Parametric-color scale 1, with time 0–0.5 sec in red and time >0.5 sec in yellow, was used to analyze tumors contrasted within 2 sec. Parametric-color scale 2 was used for tumors requiring ≥2 sec for contrast enhancement and red, cyan and yellow were used to represent the times 0-1, 1-2 and >2 sec, respectively .Following US, micro-flow imaging (MFI) was also performed using the proprietary image analysis software of the ultrasound system. The system started counting time when the contrast agent reached the tumor and the arrival time of individual pixels representing the tumor was sequentially calculated. A color map was created and automatically superimposed on a B-mode image.At-PI video images were evaluated by three ultrasonographic specialists with extensive experience  in the contrast-enhanced US of liver diseases. The specialists had no access to the clinical background of the patients, hemanalysis, imaging findings or final diagnosis. Detection of a spoke-wheel pattern was considered a positive imaging finding. Similarly, MFI images were generated to analyze contrast agent dynamics.Case 1  had a 14-mm tumor with low echo signals in hepatic segment 8 (S8). Since it took 1.7 sec to contrast the entire tumor, parametric-color scale 1 was used in At-PI analysis. Three ultrasonographic specialists identified a spoke-wheel pattern on the At-PI images, even though no identification was made in MFI.Case 2  had a 17-mm tumor with low echo signals in S8. Since 2.5 sec was required to contrast the tumor, parametric-color scale 2 was used in At-PI. Three specialists observed a spoke-wheel pattern in At-PI but not in MFI.Case 3  had a 14-mm tumor with low echo signals in S8. Since 2.8 sec was required for tumor enhancement, parametric-color scale 2 was used in At-PI. A positive diagnosis of FNH was made using MFI and At-PI.Case 4  had a 28-mm tumor with low echo signals in S5. As 2.5 sec was required for tumor enhancement, parametric-color scale 2 was used in the analysis. A positive diagnosis was made using the two imaging modalities.Case 5  had a 28-mm tumor with low echo signals in S3. Since it took 2.0 sec to contrast the tumor, parametric-color scale 1 was used in At-PI analysis. A spoke-wheel pattern was identified using MFI and At-PI. et al suggested that FNH is caused by hyperplasia of liver parenchyma in response to angiodysplasia since a number of FNH cases have involved vascular and neuroendocrine abnormalities  central scar formation, ii) nutrient vessels radiating from the center and iii) the presence of Kupffer cells . With vaSince its introduction, the ultrasound contrast agent Sonazoid has been used to thoroughly investigate the angioarchitectonic patterns of hepatic lesions whose hemodynamics were previously difficult to understand in detail –11. In aFurthermore, while it is necessary to analyze the video to fully understand contrast agent dynamics in MFI, At-PI enables the diagnosis of FNH by means of a single, static, color-mapped B-mode image that shows the direction and timing of contrast enhancement in the tumor. Therefore, it is easy to explain the findings of contrast agent dynamics, making the system highly useful in a clinical setting. We consider that At-PI is an effective tool for investigating the detailed hemodynamics of small hepatic tumors and tumors with rapid blood flow. At-PI using Sonazoid-enhanced US was useful for identifying spoke-wheel patterns of FNH <3 cm.


Carcinoembryonic antigen (CEA) in the serum and the tumour tissue of colorectal cancer (CRC) patients is the most commonly used tumour marker for the diagnosis and evaluation of prognosis or recurrence after treatment, but the role remains controversial. The objective of this study was to compare the prognostic value of CEA both in serum and tumour tissue in CRC.A total of 173 patients with CRC in stages I–III were retrospectively assessed with the endpoint of recurrence or metastasis after curative operation. CEA was assessed both in serum and tumour tissue.P=0.028) in the univariate analysis. The multivariate analysis demonstrated that the T-CEA was an independent prognosis factor in CRC. Because many factors would affect the concentration of S-CEA, there was no correlation between S-CEA and T-CEA directly.37.0% (64/173) patients had a high level of CEA in serum (S-CEA) while 39.3% (68/173) had high CEA in tumour tissue (T-CEA). There were no significant differences in clinico-pathological features between the low and high S-CEA or T-CEA groups. The high S-CEA group had a worse prognosis than the low S-CEA group but the difference was not significant. The high T-CEA group had a significantly poorer prognosis than the low T-CEA group (Our study suggests that a high T-CEA concentration may be a useful and independent predictor for poor outcome after surgery in CRC patients. It may be stronger than a high preoperative serum CEA level. Colorectal cancer (CRC) is one of the most common tumour types in the world, with about 400 000 deaths annually ,2. In thCarcinoembryonic antigen (CEA) is the most commonly used tumour marker for the diagnosis of CRC and evaluation of prognosis or recurrence after treatment. The guideline of National Comprehensive Cancer Network (NCCN) indicated that for T2 or greater lesions, a CEA test is recommended at baseline and every 3 months for 2 years . CEA canThis retrospective study included 173 patients from the database of Beijing Cancer Hospital between January 1995 and November 1999 who satisfied the following criteria: (1) sporadic CRC diagnosed in our hospital; (2) no preoperative therapy; (3) curative resection with free margin; (4) no synchronic liver or other organ metastasis detected; (5) followed up until patients had metastasis or local recurrence or until the last census point prior to closure of the study (1 August 2003); (6) staged according to the TNM system recommended by the American Joint Committee on Cancer (AJCC) [The criterions for exclusion were: (1) severe dysfunction of heart, brain, lung, kidney and liver; (2) death attributable to causes other than CRC; (3) accompanying urological or genital tumour; (4) accompanying cancers other than CRC.Five millilitres of venous blood was obtained from each patient 1 week before operation. CEA in serum (S-CEA) measurements were done by the department of clinical laboratory in our hospital using electrochemiluminescence immunoassay with Elecsys system 2010 . The cut-off value of S-CEA recommended by the manufacturers for diagnosis was 5 ng/ml. We classified the 173 patients into high S-CEA group (> 5 ng/ml) and low S-CEA group (≤ 5 ng/ml).2), deparaffinized in xylene, and rehydrated. Antigen retrieval was performed in 0.1 m citric acid buffer (pH 6.0) in a 650 W microwave for 15 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in 100% methanol for 30 min. Immunohistochemistry was performed as Power Vision™ two-step histostaining . Primary CEA antibody  was diluted as 1:50 and sections were incubated overnight at 4°C. After three washes in PBS (5 min), sections were incubated for 30 min in anti-mouse secondary antibody from a PicTure™-PV6000 Kit (Zymed Laboratories Inc.). Antibody binding was visualized using a 3,3-diaminobenzidine (DAB) kit  according to the manufacturer’s instructions.The CEA in the tumour tissue which we named T-CEA was determined by the method of immunohistochemistry. In each patient, formalin-fixed and paraffin-embedded tumour blocks were cut into 5 μm thick sections . All slides were scored by two independent and well-trained pathologists, without the knowledge of clinical and pathologic parameters or the patients’ outcomes. And in cases of scoring disagreement, a third independent assessment was performed. For all slides, at least 10 high power fields at 400× magnification were chosen randomly and > 1000 carcinoma cells were counted for each section.Stained slides were examined to identify the cellular localization of CEA immunoreactivity for both intensity  and proportion  of tumour cells stained. Integer values were assigned to the scores of intensity (0–3) and proportion of tumour cells stained (0–5). These values were multiplied together to provide a single score for each case . For T-Cspss version 10.0 . The differences in the clinico-pathological characteristics between low and high S-CEA or T-CEA expression were performed by Pearson chi-square. The relationship between S-CEA and T-CEA was also calculated by Pearson chi-square. Disease-free survival time (in months) was measured from the date of surgery to the time of event (recurrence or metastasis) or to the last census prior to closure of the study (1 August 2003). Kaplan–Meier survival analysis with the Log Rank test was used to evaluate the prognosis of serum and tissue CEA in CRC. Multivariate analysis was performed with Cox’s proportional hazard regression model to assess the effects of different variables on patients’ survival. Differences were taken as significant when P (two-tailed) was < 0.05.All statistical calculations were performed using In this study, there were 173 patients with CRC including 86 male subjects and 87 female subjects. The median age of all patients was 59 years (range 27–85 years). 37.0% (64/173) patients had a high level of S-CEA including 29 male subjects and 35 female subjects, while 63.0% (109/173) patients were in the low S-CEA group. 39.3% (68/173) patients were in the high T-CEA group .There were no significant differences in gender, age, tumour size, tumour gross type, mucin production, differentiation grade, venous invasion, stage distribution, T and N classification between the low and high S-CEA or T-CEA groups .P=0.215) (There was no significant relationship between groups of S-CEA and T-CEA (P=0.215) .P=0.3709) , but there was no significant difference between them (=0.3709) .vs 55.8 months, P=0.028)  .In order to evaluate which clinico-pathological features were independent predictors of CRC outcomes, we analysed our findings with a Cox proportional hazards model while gender, age, tumour size, histological type, differentiation grade, venous invasion, stage, S-CEA and T-CEA expressions served as covariates. Finally, four independent factors including histological type, stage, venous invasion and T-CEA were found to be significant prognostic factors for the disease-free survival of CRC. S-CEA was not found to be a significant prognostic predictor .Carcinoembryonic antigen was first described in 1965 by Gold and Freedman ,11, whenCarcinoembryonic antigen is the most widely used tumour marker worldwide and certainly the most frequently used marker in CRC. It could be detected and measured both in serum and in CRC tissue . The proIn the current study of 173 CRC patients, there was no significant difference in the disease-free survival time between low and high S-CEA. The S-CEA is not an independent prognostic factor for CRC by multivariate analysis. Previous experiments have reported that an elevated preoperative serum CEA level is a predictor for poor survival after CRC resection ,17, someet al.[The results of this series suggest that the prognostic value of T-CEA concentration may be superior to that of preoperative S-CEA level. The disease-free survival time after surgery for patients with a high T-CEA concentration was significantly shorter in univariate analysis than those with a low T-CEA. Multivariate analysis also revealed that T-CEA status (high or low) was an independent prognostic factor in CRC. The hazard of recurrence and metastasis postoperatively in the high T-CEA group is 1.587 times compared with the low T-CEA group. It is coincident with the study of Nakagoe et al.. We thinIn conclusion, our study suggests that a high T-CEA concentration may be a useful and independent predictor for poor outcome after surgery in CRC patients, and it may be stronger than a high preoperative serum CEA level.


We have investigated the structural and optical properties of type-II GaSb/InGaAs quantum dots [QDs] grown on InP (100) substrate by molecular beam epitaxy. Rectangular-shaped GaSb QDs were well developed and no nanodash-like structures which could be easily found in the InAs/InP QD system were formed. Low-temperature photoluminescence spectra show there are two peaks centered at 0.75eV and 0.76ev. The low-energy peak blueshifted with increasing excitation power is identified as the indirect transition from the InGaAs conduction band to the GaSb hole level (type-II), and the high-energy peak is identified as the direct transition (type-I) of GaSb QDs. This material system shows a promising application on quantum-dot infrared detectors and quantum-dot field-effect transistor. Quantum-size nanostructure materials have always been the research focus -5. In reHere, we provided another type-II QD material system, GaSb/InGaAs/InP, as another promising building block for optoelectronic and microelectronic applications. Compared to GaSb/GaAs type-II QDs, the bandgap of this system can be adjusted by both the QD-relevant structural characteristics and the In component in a InGaAs matrix. InP substrate is employed instead of GaAs for two reasons: one is that the lattice of InP is matched with the InGaAs buffer layer which has higher electron mobility; the other is that it may make the absorption peak position easily red shifted to 1.3 to approximately 1.55 μm. All these features show a promising application on the quantum-dot infrared detectors [QDIP] and quantum-dot field-effect transistors [QD-FET].In this work, we investigated the structural and optical properties of type-II GaSb/InGaAs QDs grown on InP (100) substrate using molecular beam epitaxy [MBE].0.53Ga0.47As buffer layer matched with the InP substrate was then deposited at a growth rate of 5,000Å/h. Four-monolayer [ML] GaSb QDs were deposited with a slow growth rate of 0.12 ML/s. There was a 3-min growth interruption before and after QD growth. Afterwards, a 30-nm In0.53Ga0.47As capping layer was grown at a rate of 5,000Å/h. A 50-nm In0.52Al0.48As barrier layer was grown at a rate of 5,300Å/h. Finally, GaSb QDs were grown for the surface morphology measurements. The growth temperature used for the whole growth process was approximately 480°C. In the growth process of the sample, the InGaAs capping layer was doped with Si.GaSb/InGaAs type-II QDs were grown on the (100) semi-insulation Fe-doped InP substrate by a V80 MBE system . The growth mode followed is the Stranski-Krastanow [SK] mode. Firstly, the surface oxides of the InP substrate were desorbed at a substrate temperature of approximately 500°C. A 500-nm InThe morphology measurements of the QDs were characterized by a atomic force microscopy [AFM] and a scanning transmission electron microscope [STEM]. The AFM measurements were conducted in a tapping mode in air, and the STEM measurements were obtained using a Tecnai F20 super-twin machine . The photoluminescence [PL] measurements were performed for the optical properties of the sample at 20 K using the source of a 532-nm line, and the excitation power is changed from 3 mW to 30 mW.0.53Ga0.47As QDs and the histogram of the height of GaSb/In0.53Ga0.47As QDs. As shown in Figure 9 cm-2 and that the shape of GaSb QDs is rectangular-shaped which is the same with GaSb/GaAs QDs  in QD-FET. In this kind of QD-FET structure, type-II GaSb/InGaAs QDs are embedded; even if the GaSb QDs directly contacts with the 2DEG, electrons will still be blocked by the GaSb barrier and will not enter the QDs. Therefore, this structure will prolong the lifetime of the holes. So, the QD-FET based on the above band structure can be used to improve the sensitivity of existing InAs/GaAs QD-FET. In addition, this material system can be fabricated on InP substrates. The higher electron mobility InGaAs light absorption layer with lattice matched to the InP substrate has a strong optical absorption in the range of 1.3 to approximately 1.55 μm which is the low-loss optical fiber window. All of these features will promote the application of QD-FET on quantum communications, night vision, and other fields. Besides, owing to the spatially separated electrons and hole characters of type-II QDs, the GaSb/InGaAs QD-based QDIP could have obviously better performance than the InAs/(In)GaAs QD-based QDIP.We have investigated the structural and optical properties of self-organized type-II GaSb/InGaAs heterostructure QDs grown on InP (100) using MBE. Formation of type-II GaSb/InGaAs heterostructure QDs centered on the PL peak at 0.75eV at 20 K. This type-II luminescence originates from radiative recombination of spatially separated electrons and holes. The PL peak positions are in proportion to the third root of the excitation power, which is a direct evidence of type-II luminescence. This structure was proposed for many important applications such as tunable laser, quantum-dot infrared detectors, and QD-FET.The authors declare that they have no competing interests.ZS participated in the MBE growth, carried out the PL measurements, and drafted the manuscript. CY conducted the STEM measurement. SZ, TH, and GH conducted the MBE growth. JH, WW, and CH coordinated the study. WL provided the idea and conceived the study together with ZL. All authors read and approved the final manuscript.


Background. The aim of this study was to investigate IL-6 and 25-hydroxyvitamin D (25-OH D) associations with muscle size and muscle function in dialysis patients. Methods. Patients were included in a 16-week control period followed by 16 weeks of high-intensity resistance training thrice weekly. IL-6 and 25-OH D were analysed after an over-night fast. Muscle fibre size was analysed in biopsies from  m. vastus lateralis. Muscle power was tested using a Leg Extensor Power Rig.  Results. Patients (n = 36) with IL-6 ≥ 6.49 pg/ml (median) were older and had decreased muscle power and a reduced protein intake (P < 0.05) compared with patients with IL-6 < 6.49 pg/ml. IL-6 was not associated with muscle fibre size. Vitamin D deficiency (25-OH D < 50 nmol/l) was present in 51% of the patients and not associated with muscle power. IL-6 remained unchanged during the training period, whilst muscle power increased by 20–23% (P < 0.001).  Conclusion. Elevated IL-6 values were associated with decreased muscle power but not with decreased muscle fibre size. Half of the patients were suffering from vitamin D deficiency, which was not associated with muscle power. IL-6 was unchanged by high-intensity resistance training in dialysis patients in this study. Chronic kidney disease (CKD) can arise as the result of several diseases, among them diabetes. When renal function is reduced to approximately 10% of the healthy renal capacity of an individual, dialysis therapy or kidney transplantation is needed in order to keep the patient alive. The majority of patients with CKD are treated with haemodialysis thrice weekly or daily by means of peritoneal dialysis. per se, or both [Low-grade systemic inflammation with increased levels of proinflammatory cytokines is common in patients with CKD . The ori or both . The low or both , 4. WhilIn subjects not suffering from CKD, low-grade inflammation is associated with physical inactivity . As physIn parallel with low-grade inflammation, vitamin D deficiency determined as low values of 25-hydroxyvitamin D (25-OH D) is common in patients with CKD –8. The vThus, low-grade inflammation and vitamin D deficiency may have a negative impact on muscle mass and muscle function in dialysis patients. The aim of this study was to investigate the effect of resistance training on the proinflammatory factor IL-6 as well as the impact of 25-OH D on muscle size in dialysis patients.The data from the present study were obtained as part of a recent study of effects of resistance training in dialysis patients . The patComorbidity level was assessed using the Index of Coexistent Disease . Food incontrolled-trials.com no. ISRCTN72099857.The tests were conducted with the same relation to the dialysis schedule at the three tests. Informed consent was obtained from all patients and the local ethical committee approved the protocol (H-D-2008-124). The study was registered on The training consisted of supervised progressive high-intensity resistance training three times a week for 16 weeks . The traWhilst the predefined number of repetitions during every training session was completed in the first two to three sets, the number of repetitions usually declined in the following sets as exhaustion was achieved earlier.Leg extensor power was measured using the Nottingham Leg Extensor Power Rig. The patients sat in an upright position with their arms folded across their chest and with their active leg towards the push-pedal in front of the seat, which made the direction of movement almost horizontal. The subject was instructed to push the pedal as hard and as fast as possible. The measurement was repeated at least five times and until no further improvement could be recorded on two consecutive occasions. The data were recorded, computed, and expressed in watts (W) using the Leg Rig software package .Physical function was measured using the Chair Stand Test from the Senior Fitness Test . The tes m. vastus lateralis on nonhaemodialysis days through a five-millimetre incision using a Bergström biopsy needle. Serial sections (10 μm) of the muscle biopsy samples were cut and myofibrillar ATPase histochemistry was performed at pH 9.40 after preincubation at pH values 4.37, 4.60, and 10.30 [Muscle biopsies were obtained from the midregion ofnd 10.30 . Computend 10.30 . Fibre t2 D), and 25-OH D were analysed from blood taken after an overnight fast and a minimum of 18 hours after the last training session.Interleukin-6, 1,25-dihydroxyvitamin D . According to the manufacturer, the limit of detection for this kit was 1.5 pg/mL, and the kit has been found to show no cross-reaction with such substances as IL-1Serum 25-OH D was analysed using an immunological method that measures both vitamin D 3 and D 2 . Analyse t-test was used to test for differences between the baseline test and pretraining and between pre- and posttraining and also to compare the differences during the control period with any difference during the training period. The Mann-Whitney Test or unpaired Student's t-test was used to compare groups of patients. Age adjustments were performed using linear and logistic regression, and entered analyses and variables were log-transformed if the residuals were not normally distributed. Data are presented as the mean ± standard error of the mean (SEM), count, or percentages. All tests were two-tailed at a significance level of P ≤ 0.05.Data distributions were tested using a Shapiro-Wilk Test. In analyses of variables with nonnormal data distributions nonparametric statistics were used. Binary correlations were tested using the Spearman Test. The Wilcoxon Signed Ranks Test or paired Student'sThirty-six patients were included and 23 patients completed the intervention. Six patients dropped out during the control period and three patients dropped out during the training period due to problems not related to training. Four patients completed the intervention but were not retested due to illness, change of dialysis modality, or problems arising during blood sampling. Two patients were not included in the IL-6 measurements and one patient was not included in the 25-OH D measurements owing to insufficient plasma collection.P = 0.290). Interleukin-6 was positively correlated with age  and CRP . At baseline, 51% of the patients were vitamin D deficient. There was no gender difference for 25-OH D .Clinical characteristics are presented in −1·kilo−1 body weight, and had impaired muscle power compared to those with reduced IL-6 values. When muscle power was age and gender adjusted, low muscle power in the left leg remained associated with elevated IL-6 values (P = 0.042), whereas impaired muscle power in the right leg tended to be associated with elevated IL-6 values (P = 0.082).Clinical data in patients with reduced or elevated IL-6 values (split on the median 6.49 pg/mL) are presented in Mean IL-6 remained stable throughout the control and training period . When thThe IL-6 values tested prior to and during the training period did not correlate with changes in muscle power or muscle morphology during the training period (data not shown).P = 0.012) and a reduction in type 1 muscle fibre size (P = 0.013).Plasma albumin was elevated in patients with normal 25-OH D compared to those with vitamin D deficiency . Patient25-OH D was decreased after the training period . In Figur = −0.582, P = 0.009).The 25-OH D status before and during the training period was not associated with changes in muscle power or muscle morphology during the training period (data not shown). However, decreased 25-OH D during the training period was correlated with an increase in the size of type 2 muscle fibres  [P = 0.164) and increased during the training period  [μm2 and type 2: 3185 ± 233 μm2 versus 3606 ± 275 μm2, resp.). Body weight increased during the training period from 72.8 ± 3.7 to 73.9 ± 3.8 kg, P = 0.05.As previously shown, muscle power remained unchanged during the control period and increased during the training period  . Physica< 0.001) . Muscle The main finding in this study was that 16 weeks of resistance training did not affect the level of the circulating proinflammatory cytokine IL-6 in a well-nourished sample of dialysis patients with relatively high values of plasma albumin. In cross-sectional analyses an elevated level of IL-6 was associated with impaired muscle power and a reduced protein intake. In addition, half of the patients were found to be suffering from vitamin D deficiency. per se [The IL-6 mean value was comparable with values reported in other studies focusing on skeletal muscle and exercise training in dialysis patients , 22. In  per se , insulin per se , anaboli per se , and aci per se  are factThe literature reports that exercise training may decrease elevated levels of circulating IL-6 in subjects without CKD , 28, 29 It is well documented that exercise induces acute increased levels of circulating IL-6, which is stimulated by contracting muscle , 28. IntVitamin D values were in line with data reported in other studies on dialysis patients , 9, 34. Vitamin D deficiency in CKD patients is treated to prevent secondary hyperparathyroidism and osteomalacia. In addition, treatment with 1,25-dihydroxyvitamin D has in a retrospective cross-sectional study been suggested to be associated with greater muscle size and muscle strength in dialysis patients . HoweverThese results are limited, however, by the relatively low number of patients. In relation to the data for muscle size, estimations of muscle mass in the lower extremities determined by scanning methods would have been relevant in addition to a measure of muscle size at the fibre level. Scanning methods could have provided additional information of whole leg muscle mass and intramuscular fat content as relevant outcomes in this study.In conclusion, in a well-nourished sample of dialysis patients, elevated serum IL-6 values were not decreased by a period of resistance training. Half of the patients were found to be suffering from vitamin D deficiency. The low-grade inflammation was associated with reduced muscle power but not with muscle fibre size. Elevated IL-6 values and vitamin D deficiency did not affect the positive effects of resistance training on muscle power and physical function.


H) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (V H and VL domains of the antibodies with a short polypeptide linker. scFv specific to any particular antigen may be easily generated by Phage display  is a fusion protein formed by engineering the association of the VE. coli, Yeast, mammalian, insect cell, wheat germ cell-free expression system, and plant-based expression system , was used for the construction and expression of two target genes in an appropriate host strain. By co-expression of a periplasmic chaperone (Skp) with scFv, a relatively high amount of soluble scFv antibody was successfully obtained.V. parahaemolyticusCGMCC1.2164, V. parahaemolyticusCGMCC1.1614, V. parahaemolyticusCGMCC1.1615, V. parahaemolyticusCGMCC1.1616 were purchased from the Institute of Microbiology, Chinese Academy of Sciences . All other strains were from Fujian Agriculture and Forestry University . The plasmids pET28a(+) and pET32a(+) were purchased from Novagen , and the vector pACYC-Duet-1 was generously provided by Professor Lijun Bi . The vector pGEPi was generously provided by Dr Mengfei Ho . DNA restriction enzymes were purchased from Promega (USA). Taq DNA polymerase and T4 DNA ligase were purchased from Takara . Anti 6×His tag monoclonal antibody was purchased from Abgent (USA), and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was from Boster Biological Technology Co. . All oligonucleotides were listed in Table V. parahaemolyticus(CGMCC1.2164) genome was used as a template to amplify the tlh gene[GeneBank:GU971665.1] . Expression of the target protein was induced by adding 1 mM IPTG when the culture reached an OD600 of 0.8, and the induced cells were grown at 37°C for overnight, and then harvested by centrifugation. After SDS-PAGE, the expressed proteins were stained using 0.3 M CuCl2 by shaking for 5 min at 37°C, and visualized against a black background. The target TLH protein bands were cut and mashed, and mixed with 1 mL PBS. The mixture solution was dialyzed with Gly/Tris buffer and the supernatant was harvested by centrifugation, and the extracted supernatant containing the TLH protein was visualized by SDS-PAGE. Protein concentration was determined by the BCA protein assay kit.The skp was first constructed. The DNA skp gene[GeneBank:U00096.2] encoding the molecular chaperone was amplified using the primers Skp-F and Skp-R, and JM109 genomic DNA was used as template. The amplified DNA fragment was digested by Bgl II and Xho I, and ligated the pACYC-Duet vector digested with the same restriction enzymes. To construct four different formats of expression vectors, the segment encoding anti-TLH scFv was amplified using the template vector pCANTAB-5E-scFv  and pET32a(+) vectors digested with EcoR I and Hind III, and the resultant vectors were designated as pET28a-scFv and pET32a-scFv, respectively. The amplified scFv-2 fragment was digested with EcoR I and Hind III, and ligated with pACYC-Duet and pACYC-Duet-skp vectors digested with the EcoR I and Hind III, and the resultant vectors were designated as pACYC-Duet-scFv and pACYC-Duet-scFv-skp, respectively.pET28a(+), pET32a(+), and pACYC-Duet vectors were used for the construction of expression vectors with the scFv proteins fused to 6×His tag, 6×His-TRX, and 6×His tag, respectively. For co-expression, a recombinant vector pACYC-Duet-E. coli BL21 by electroporation, and a single colony from the selection plate was inoculated into 5 mL LB liquid media containing either 100 μ g/mL ampicillin or 50 μ g/mL kanamycin or 34 μ g/mL chloramphenicol. The culture was incubated overnight with shaking at 37°C, and then transferred to a larger-scale LB media (1 mL culture transferred into 500 mL fresh LB). Expression of the target protein was induced by adding 1 mM IPTG when the culture reached an OD600 of 0.8. Cells were grown for an additional 12 or 32 h (only for pACYC-Duet-scFv-skp/BL21) at 16°C, and then harvested by centrifugation. To ensure the accuracy of the experiment, four different derived cultures were adjusted to the same concentration for samples treatment, and the treated proteins were analyzed by SDS-PAGE using 12% (v/v) polyacrylamide gels.For protein expression, the recombinant plasmids were transformed into 2+ affinity chromatography. The expressed product was first harvested by centrifugation (10000 r/min) at 4°C for 10 min, followed by addition of 5 mL of the binding buffer  after the removal of the supernatant. The complex was then mixed gently, sonicated and then centrifuged at 4°C for 10 min. The supernatant was collected and loaded onto a 3 mL Ni2+-NTA column, which was equilibrated with the binding buffer before the purification. After allowing the sample to flow-through, the column was washed twice with the wash buffer (100 mL/time), and the wash fraction was collected for SDS-PAGE analysis. After the wash, the retained protein of interest was eluted with 0.5 mL elution buffer 4 times, and the eluate was collected and analyzed by SDS-PAGE and verified by Western blotting. Protein concentration was determined by the BCA protein assay kit.The purification of the expressed anti-TLH scFv was performed using Ni2SO4. Absorbance at 450 nm was measured using a microplate reader.The activity of the purified scFv products was determined by ELISA. The dialyzed TLH antigen (without 6×His tag) was used to coat the 96 well plates at 4°C for overnight. After blocking and washing the ELISA plate, the purified scFv products derived from four different formats of plasmids were added to the reaction wells and incubated at 37°C for 2 h. Then, the anti-His tag antibody was added to the reaction wells and incubated at 37°C for 2 h. The binding activity of the purified scFv was detected by using a HRP conjugated anti-mouse IgG antibody. The enzyme reaction was then performed with TMB as a substrate and color development was terminated with 2 M HV. parahaemolyticus strains, seven Vibrio strains from other species, and four non-Vibrio bacterial strains were tested using ELISA. The bacterial cultures were grown in 5 mL APW medium without shaking at 37°C overnight, and then their supernatants were collected by centrifugation . The supernatants were added to a 96 well plate (100 μL/well) to perform ELISA as described before.Similarly, ELISA was also used for the detection of bacterial TLH. Totally, eleven different types of To further confirm the binding activity of scFv (co-expression), western blotting was performed as described by Singh et al.  with minV. parahaemolyticus antigens (Vp1668 and YscF) were coated on 96-well plates  at 4°C for overnight, respectively. After the blocking and washing, the purified scFv (pACYC-Duet-scFv-skp) was added to the reaction wells and incubated at 37°C for 2 h. Then the anti-His tag antibody was added to the reaction wells and incubated at 37°C for 2 h. The specificity of the scFv was detected with the HRP conjugated anti-mouse IgG antibody.ELISA was performed to determine the specificity of the expressed anti-TLH scFv. BSA, KLH, and associated The bio-molecular interaction and affinity of the scFv to TLH was measured using a TA-instrument Nano ITC-LV. The measurement was automatically monitored by data acquisition software supplied along with the instrument. All the measurements were carried out at 25°C. The affinity constant was calculated using Origin software supplied with the Nano ITC-LV.scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv were constructed  in E. coli BL21, with the expressed scFv contains a 6×His tag at C-terminal. The expression vector pET28a-scFv encodes scFv with two 6×His tags, one each at the N- and C-terminal, respectively, while the expression vector pET32a-scFv encodes a TRX-scFv fusion protein with two 6×His tags, one each at the N-terminal and C-terminal, respectively.In this study, four different expression vectors pACYC-Duet-d Figure , and tra2, and visualized against a black background. The target TLH protein bands were cut and mashed with PBS buffer. After harvested by centrifugation, the extracted supernatant containing the TLH protein was visualized by SDS-PAGE. As shown in Figure After the TLH protein was expressed, the total protein was analyzed by SDS-PAGE, and the gel was stained using 0.3 M CuClE. coli BL21 (DE3) by electroporation for the expression of their respective target proteins. The transformants were grown in test-tube cultures and induced with 0.5 mM IPTG at 16°C for 12 or 36 h, and the expressed products were analyzed by SDS-PAGE. As shown in Figure Four constructed vectors were used to transform 600 value. As shown in Figure 600  polyacrylamide gels. The concentrations of purified proteins from four different constructs are 0.13, 0.29, 0.115, and 0.38 mg/mL, respectively. After SDS-PAGE analysis, the target protein was further transferred onto a PVDF membrane, and anti-6×His tag antibody was used to confirm the presence of the expressed scFv protein. Protein purification and western blotting results showed that all four different fusion proteins were purified successfully  were tested. Table V. parahaemolyticus strains were successfully detected by Skp co-expressed scFv. In contrast, seven Vibrio strains from other species and four other bacterial strains of non-Vibrio all gave negative detection results. These results showed that this Skp co-expressed scFv could specifically recognize TLH positive V. parahaemolyticus strains, and might be used as a potential reagent for V. parahaemolyticus diagnosis.We were interested in determining whether the co-expressed scFv could serve as a good antibody reagent to directly detect TLH from bacterial strains. In the first detection, three TLH positive Although scFv have been used as therapeutic agents and plays a useful role in diagnosis of diseases . This improvement of solubility can be attributed to the following three advantages of lower expression temperature: (1) The expression rate of protein is greatly reduced, leading to enhanced the folding efficiency of protein; (2) The endogenous proteases are usually inactive or have low-activity, leading to an enhancement of target protein stability (Subedi et al., Low temperature is an important factor in soluble protein expression (Qing et al., skp vector system is an effective system for soluble expression of scFv.We constructed four different expression vectors, and tested their ability to express the soluble scFv protein. The solubility and binding activity of the purified protein were analyzed by SDS-PAGE and ELISA, respectively. Amongst the four purified protein, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3–4 fold higher than other three purified scFv. Also, the scFv obtained by Skp co-expression was specific for TLH and did not cross-react with other TLH-associated proteins. Notably, this scFv could be used to detect TLH directly in real samples, suggesting that the pACYC-Duet-Rongzhi Wang, Youjun Feng, and Shihua Wang designed the experiments and wrote the manuscript. Rongzhi Wang, Shuangshuang Xiang, Yonghui Zhang performed all the experiments. DL Wu, Youjun Feng, Swaminath Srinivas, and Mingshen Lin performed a few experiments and data analysis. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Augment is a bone graft substitute intended to be used as an alternative to autologous bone graft in the fusion of hindfoot and ankle joints. Nonclinical studies included assessment of the pharmacokinetic profile of intravenously administered recombinant human platelet-derived growth factor-BB in rat and dog, effects of intravenous administration of recombinant human platelet-derived growth factor-BB in a reproductive and development toxicity study in rats, and chronic toxicity and carcinogenicity of Augment in a 12-month implantation model. These studies showed that systemic exposure was brief and clearance was rapid. No signs of toxicity, carcinogenicity, or tumor promotion were observed even with doses far exceeding the maximum clinical dose. Results of clinical trials (605 participants) and commercial use of recombinant human platelet-derived growth factor-BB containing products indicate that these products are not associated with increased incidence of adverse events or cancer. The safety data presented provide evidence that recombinant human platelet-derived growth factor-BB is a safe therapeutic when used in combination products as a single administration during surgical procedures for bone repair and fusion. There is no evidence associating use of recombinant human platelet-derived growth factor-BB in Augment with chronic toxicity, carcinogenicity, or tumor promotion.This article discusses nonclinical and clinical data regarding the safety of recombinant human platelet-derived growth factor-BB as a component of the Augment While these isoforms have different binding specificities to PDGF cell-surface receptors, PDGF-BB is considered the universal PDGF isoform because it can bind to all known PDGF receptors.3 PDGF-BB is a primary component of platelets released at sites of injury following platelet activation. PDGF-BB is chemotactic and mitogenic for cells of mesenchymal origin.3 Additionally, PDGF-BB is proangiogenic, upregulating vascular endothelial growth factor (VEGF) to stimulate new capillary growth.2 Through these biological activities, PDGF-BB contributes to tissue repair.The platelet-derived growth factor (PDGF) family contains five members found naturally in the body. These are AA homodimer, AB heterodimer, BB homodimer, CC homodimer, and DD homodimer.® . Later efforts concentrated on regeneration of periodontal bone with GEM 21S® . Nonclinical orthopedic studies, including fracture repair in models of impaired healing (geriatric/osteoporotic or diabetic),5 distraction osteogenesis,6 and spine fusion7 demonstrated the efficacy of rhPDGF-BB in bone augmentation and the foundation for development of clinical applications.12The development of recombinant human PDGF-BB (rhPDGF-BB) for therapeutic purposes focused initially on soft tissue healing with Regranex® Bone Graft (Augment) comprises an osteoconductive scaffold of β-tricalcium phosphate (β-TCP) and rhPDGF-BB. rhPDGF-BB is a protein of approximately 24.5 kDa produced using recombinant DNA technology in a Saccharomyces cerevisiae expression system. The two components are mixed prior to implantation. Augment is intended as an alternative to autologous bone graft (autograft) in hindfoot or ankle joint fusion sparing the patient the morbidity of autograft harvest. The average dose of rhPDGF-BB administered in orthopedic clinical trials using Augment is 1.8 mg (6 cm3 of β-TCP + 6 mL of 0.3 mg/mL rhPDGF-BB); the maximum clinical dose is 2.7 mg (9 cm3 of β-TCP + 9 mL of 0.3 mg/mL rhPDGF-BB).Augment14 However, as discussed below, the emerging data appear to provide reassurance about the safety of rhPDGF-BB. This is, in part, due to the fundamental differences between the local single administration of rhPDGF-BB in a combination device such as GEM 21S or Agument and the continuous deregulated expression of PDGF-BB in certain cancers.Due to its biological activity, altered expression of PDGF-BB has been associated with concerns of potential tumor promotion.Here, we present the latest nonclinical studies and review the data on the safety of rhPDGF-BB.Testing was conducted in accordance with guidance from the International Organization for Standardization (ISO), International Conference on Harmonisation (ICH), and United States Pharmacopeia (USP). Animal studies were performed at facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) under protocols approved by the Institutional Animal Care and Use Committee (IACUC) review . rhPDGF-® .Two studies were carried out to evaluate circulating levels of rhPDGF-BB administered as a single intravenous (IV) dose. In the first study, 48 Sprague Dawley rats were distributed into two groups that received a single IV dose of either 0.44 mg/kg rhPDGF-BB or 20 mM sodium acetate. Serum samples were collected at baseline, 1, 5, 10, and 20 min and 1, 4, 8, 24, 48, 72, 96, and 168 h post dose (n = 6 per group per time point). In the second study, eight beagle dogs were distributed into two groups that received a single IV bolus dose of either 1.0 mg/kg of rhPDGF-BB or 20 mM sodium acetate. Samples were collected at the same time points listed above (n = 4 per group per time point). For both studies, the concentration of rhPDGF-BB was determined by enzyme-linked immunosorbent assay (ELISA). Pharmacokinetic analyses were performed using a noncompartmental module of WinNonlinThe potential toxicity of rhPDGF-BB was studied by daily IV administration in gravid rats over 21 days of gestation. Three groups of 22 female rats received single daily doses of vehicle control  or rhPDGF-BB at a dose of either 40 or 400 µg/kg/day via IV injection for 21 days. On day 21 of gestation, all rats were euthanized and a gross pathological examination was performed. The reproductive tract of each dam was dissected, the ovaries were removed, and the corpora lutea were counted. The gravid uterus was weighed, the uterine contents were examined, and the number and position of live and dead fetuses were recorded. Each fetus was weighed and given a detailed external examination. Additional groups of six rats per group were included for toxicokinetic analysis at baseline and day 21. Serum rhPDGF-BB concentration and antibody formation against rhPDGF-BB were determined using ELISA.In all, 300  Sprague Dawley rats were distributed into three groups. β-TCP was combined with either 0.3 mg/mL of rhPDGF-BB (test) or sodium acetate (control). Aliquots of 200 µL of test or control article were implanted adjacent to the femur, underneath the muscle; the third group received sham surgery. Animals were euthanized after 30 (n = 10 per group per sex), 180 (n = 10 per group per sex), or 365 days (n = 30 per group per sex). Macroscopic and microscopic evaluations of toxicity and tumor incidence were performed. Serum was collected for hematology and clinical chemistry determinations. Anti–PDGF-BB antibody formation was evaluated by ELISA.max was observed at 1 min with a Cmax of 6161.2 ng/mL. The Area under the curve from time 0 to 4 hours AUC0–4 was 380.7 h × ng/mL, and the clearance was 17.5 mL/min/kg  occurred 1 min post dose. Half-life was 0.92 h. Inferred Area under the curve AUCinf was 2504.4 h × ng/mL, and the clearance was 423.3 mL/min/kg . The TL/min/kg . FollowiL/min/kg .No treatment-related mortality or significant adverse effects were observed. Effects on body weight, body weight gains, and food consumption were unremarkable. The gross pathological assessment was unremarkable in all treatment groups. The uterine parameters assessed  were unaffected in all treatment groups. Fetal weights were unaffected by treatment. The incidence of litters and fetuses with major malformations was unaffected by the treatment across all treatment groups. The incidence of minor external and visceral anomalies was unaffected by rhPDGF-BB. Rat plasma samples were assayed for rhPDGF-BB using an ELISA. The plasma levels of rhPDGF-BB in all dams and fetuses were below the level of detection (<0.625 mg/mL). No anti–rhPDGF-BB antibodies were detected in the 45 samples tested except in one pretreatment sample of one of the dams.No treatment-related mortality or effects on the clinical condition of the rats were observed. No remarkable test article–related changes in the body weight or body weight gain were observed. No significant changes in urinalysis parameters across treatment groups and gender were observed. Similarly, no significant changes in bone marrow parameters across treatment groups and gender were observed. There were no test article–related microscopic findings on days 30, 180, or 365 of the study. On day 30, minimal foreign body granulomas containing material consistent with surgical sutures were present at the implant site across groups. Minimal to mild granulation tissue was noted at the implant site in animals from the control and the test article groups on day 30, day 180, and day 365. Because this was observed in both groups, it was likely a local reaction to the β-TCP. There were no test article–related neoplastic microscopic observations noted in either sex on day 365. None of the animals treated with the test article were positive for anti–PDGF-BB antibodies.16 Repeated daily administration of rhPDGF-BB by IV injection throughout the gestation period did not have any adverse effect in the dams or their progeny. A 1-year toxicity study demonstrated that implantation of rhPDGF-BB in combination with β-TCP did not elicit any adverse effects, tumor formation, or untoward immune reaction against the device. These outcomes highlight the safety of Augment, which is an implantable combination product intended for a single local administration for orthopedic tissue repair.The safety of rhPDGF-BB alone or in combination with β-TCP has been demonstrated in a comprehensive battery of in vivo and in vitro studies. The test materials were not mutagenic, hemolytic, cytotoxic, pyrogenic, or allergenic, and there was no evidence of either local or systemic toxicity.19 Characterization of the interaction of PDGF with plasma proteins determined that this plasma protein was α2-macroglobulin.19 The α2-macroglobulin is present in two forms: a native and a transformed conformation, which occurs after protease activation. The receptor-mediated effects of PDGF (chemotaxis and mitogenesis) are inhibited by binding to α2-macroglobulin.18 Interaction with α2-macroglobulin also regulates the clearance of PDGF. PDGF binds preferentially to the conformationally transformed α2-macroglobulin,20 leading to rapid clearance of the complex through the low-density-lipoprotein-receptor-related protein (LRP) pathway.21 Bonner et al.22 proposed that native α2-macroglobulin binds PDGF, serving as a reservoir of PDGF to be released in the healing environment (i.e. sites of injury with low pH) but keeps it sequestered from systemic exposure. The inflammatory response and presence of proteases in sites of injury increase the conformationally transformed α2-macroglobulin leading to preferential binding of PDGF and rapid clearance from the circulation.PDGF is released from platelets at sites of injury and has a localized stimulatory effect on the wound healing process. Intrinsic clearance mechanisms limit systemic availability and regulate the local activity of PDGF. The presence of a plasma PDGF–binding protein was described in 1984.125I-rhPDGF-BB) demonstrated that 60% to 70% of the locally administered rhPDGF-BB was released from the implantation site in the first 60 min, followed by a slower, sustained release over several days.16 An additional study to assess the pharmacokinetics differences between IV administration of 125I-rhPDGF-BB and intramuscular (IM) implantation of 125I-rhPDGF-BB combined with β-TCP determined that the systemic bioavailability was similar by both routes of administration.16 Two studies with nonlabeled rhPDGF-BB delivered via IV administration to rats and dogs corroborate the findings of the studies performed with isotope-labeled rhPDGF-BB. In these studies, determination of rhPDGF-BB levels in serum was performed by ELISA. The half-life, Tmax, Cmax, and clearance were comparable to those reported in previous studies.16The results of the in vivo studies using radio-labeled rhPDGF-BB  results in reversal of the transformation.30 This suggests that these tumors are PDGF-BB dependent, requiring continuous exposure to PDGF-BB to maintain their transformed state. Furthermore, these observations may reflect autocrine or paracrine stimulation of proliferation rather than deregulation of the cell cycle that is the hallmark of transformed cells.31PDGF-B shares sequence homology with the simian sarcoma virus oncogene, 32 This is reinforced by the data from a multicenter, randomized, controlled clinical trial comparing Augment and autograft (2:1 randomization scheme) with 414 study subjects and a 12-month follow-up. Five cancer events were reported in this clinical trial: three in the Augment group and two in the autograft group ; none of the tumors in the Augment group were at or near the site of implantation  found no increased risk of cancer incidence or cancer mortality with administration of rhPDGF-BB.antation . The canAugment. Upon review of all the available information from more than 150,000 implanted units, only one potential cancer-related event has been reported. The only commercial use of Augment has been in Canada where the product was approved in 2009 for foot and ankle fusions. Between 2009 and 2011 there were over 200 kits of Augment sold without receipt of a single report of adverse events (including cancer).GEM 21S, which was approved by the Food and Drug Administration (FDA) periodontal bone regeneration, is essentially identical to Augment has been evaluated for potential toxicity in a variety of studies without suggestion of toxicity, carcinogenicity, or mutagenicity after a single administration as an implantable device/drug combination product. The biological activity of rhPDGF-BB is regulated by α2-macroglobulin resulting in brief systemic exposure and rapid elimination of the protein by normal metabolic routes. Data from multiple clinical trials with Augment and GEM 21S and use of GEM 21S in clinical practice indicate that cancer incidence is not increased with clinical application of rhPDGF-BB.The use of rhPDGF-BB and


Phase-contrast magnetic resonance imaging is a technique that allows for characterization of regional cardiac function and for measuring transmural myocardial velocities in human hearts with high temporal and spatial resolution. The application of this technique  to murine hearts has been very limited so far. The aim of our study was to implement and to optimize tissue phase mapping for a comprehensive assessment of murine transmural wall motion. Baseline values for regional motion patterns in mouse hearts, based on the clinically used American Heart Association's 17-segment model, were established, and a detailed motion analysis of mouse heart for the entire cardiac cycle  is provided. Black-blood contrast was found to be essential to obtain reproducible velocity encoding. Tissue phase mapping of the mouse heart permits the detailed assessment of regional myocardial velocities. While a proof-of-principle application in a murine ischemia–reperfusion model was performed, future studies are warranted to assess its potential for the investigation of systolic and diastolic functions in genetically and surgically manipulated mouse models of human heart disease. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc. Genetically and surgically modified mice have become the animal models of choice for human cardiac disease. Fast, high-resolution, multiframe magnetic resonance imaging (cine-MRI) has been established in recent years as a routine tool in many research laboratories around the world to noninvasively assess global cardiac function –6. Left This article aims to overcome the limitations described above. Specifically, we sought: (A) to investigate the feasibility of a comprehensive assessment of myocardial wall motion in three dimensions analogously to clinical practice at three levels in the heart , using a conventional, commonly available preclinical MR system interfaced to a 9.4-T magnet; (B) to optimize the method with respect to temporal/spatial resolution and scan time; (C) to demonstrate the benefits of black-blood over bright-blood contrast in murine phase-contrast MRI; (D) to establish baseline values for regional motion patterns in mouse hearts, based on the American Heart Association 17-segment model ; (E) to All MR experiments were carried out on a 9.4-T (400 MHz) MR system  comprising a horizontal magnet (bore size = 210 mm), a VNMRS Direct Drive™ console, an actively shielded gradient system , and a quadrature-driven birdcage resonator .n = 22, 23.1 ± 1.8 g) were obtained from a commercial breeder  at least 1 week before the first imaging time point to allow naturalization to new surroundings. The mice were kept under controlled conditions for temperature, humidity, and light, with chow and water available ad libitum. Anesthesia was induced in an anesthetic chamber using 4% isoflurane in 100% oxygen. Animals were then positioned prone on dedicated mouse cradles and maintained at 1.5–2% isoflurane at 2 L/min oxygen flow throughout the MRI experiments. A 0.5-mL Eppendorff tube filled with 1% agarose (spiked with 2 mM Gd-DTPA) was placed next to the chest of the animal to correct background phase errors , the left coronary artery was occluded for 45 min followed by reperfusion as described in Ref.All investigations conformed to Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 (HMSO) and to institutional guidelines.2, matrix size = 128 × 128, TE/TR = 2.1/4.6 ms, slice thickness = 1 mm, 15° sinc excitation pulse, number of averages NT = 2, vencin-plane = 6 cm/s, and vencthrough-plane = 8 cm/s. Velocity-encoded/compensated scans were acquired in an interleaved fashion. For each line in k-space, the motion-compensated acquisition was executed for all time frames of one cardiac cycle, followed by the three motion-encoded scans in three successive heartbeats before incrementing the phase-encoding gradient . A saturation module  at the end of each cine-train, which consisted of two 4-mm saturation slices, applied 4.5 mm above and below the imaging slice, followed by a crusher gradient, provided black-blood contrast. The remaining ventricle was covered with conventional bright-blood cine-imaging by turning off the motion-encoding and black-blood modules . All sequences were ECG-triggered and respiratory-gated with steady-state maintenance during respiration [i.e., the sequence continued to run during respiration with the same timing , but no data were acquired, and all internal counters were not incremented—see Ref.The phase-contrast imaging sequence was based on a two-dimensional multiframe gradient echo sequence. Motion-compensating and -encoding gradients were combined with the imaging gradients as reported previously , 17. AftVelocity assessment by means of phase-contrast MRI was validated on a flow phantom with pulsatile flow using an MR-compatible flow-sensor (placed inside the magnet and outside the RF coil) before in vivo application. TPM experiments were conducted at three different flow rates  with venc's of 40, 50, and 60 cm/s, respectively. A trigger signal was derived from the flow curve and used to synchronize the MR experiment.n = 6, 23.6 ± 1.2 g), where a mid-ventricular slice was additionally acquired with identical sequence parameters, but the saturation module at the end of the cine-train turned off. Typically, two more frames were obtained instead in this case. To exclude the inherent variability of the black-blood contrast technique, its reproducibility was assessed in three more mice (31 ± 4 g) by repeating the acquisition of a mid-ventricular slice with black-blood contrast.The benefit of black-blood over bright-blood contrast was investigated in a subset of mice . All raw data were isotropically zerofilled by a factor of two and filtered (modified third-order Butterworth filter (25)) before Fourier transformation resulting in a reconstructed in-plane voxel size of 100 × 100 μm2.Reconstruction and analysis of data were performed offline using purpose-written x-, y-, and z-directions were obtained by subtracting the respective motion-encoded scan from the phase reference . Magnitude images (for segmentation) were calculated by adding the magnitude of all four scans. Phase maps and magnitude data were exported into binary format and read by a purpose-written software tool to analyze the TPM data in MATLAB . After manually delineating the epicardial and endocardial borders on a frame-by-frame and slice-by slice basis, the measured in-plane velocities (vx and vy) were transferred into an internal polar coordinate system, positioned at the centre of mass of the left ventricle, and expressed as radial (vr) and circumferential  velocities. Furthermore, all velocities were corrected for bulk motion by subtracting global translational velocities from the local velocity components  as:The sign convention for the various velocities used throughout the article follows Ref.t-test. A P-value of 0.05 was considered statistically significant. Furthermore, correlation coefficients were calculated by correlating mean radial velocities in 24 angular segments with the global velocity time course  regression and (2) Bland–Altman analysis using Microsoft Excel 2008 for Apple MacIntosh. The total number of data points available for both analyses is equal to the sum over all time frames and animals. The null hypothesis that the slope in the regression analysis was equal to one was tested using a Student's e course .For each data set of the normal hearts, cardiac structural  and functional parameters were obtained from TIFF images as described before (5), using Amira 4.1 .The flow rates in the validation experiment were quantified to 35.3, 40.6, and 44.7 mL/min and were therefore in excellent agreement with the preset flow rates. The accuracy of the flow rate at the pump was ±0.5 mL/min, whereas the standard deviation, derived from the phase noise in the experiments, for the flow measured with phase-contrast MRI was 0.01 mL/min.Linear regression analysis was used to assess the agreement between velocities obtained by black-blood versus bright-blood methodologies and to assess reproducibility of the black-blood data following repeated acquisitions. Comparisons were made by calculating mean velocities averaged over the entire LV segmentation mask for all time frames, and the slopes and correlation coefficients are listed in Excellent reproducibility for these data was also confirmed by the Bland–Altman-analysis shown in Of the 22 mice scanned, one was excluded due to gradient interference with the ECG and subsequent mistriggering. vφ describes the rotational motion .Global cardiac functional parameters were within published values for normal mice. Particularly, we measured for: LV-mass: 74.1 ± 4.6 mg, end-diastolic volume: 45.8 ± 6.9 μL, end-systolic volume: 12.6 ± 4.9 μL, SV: 33.2 ± 5.2 μL, ejection fraction: 72.9 ± 8.2%, heart rate 468 ± 33 bpm, and cardiac output: 15.5 ± 2.4 mL/min  DENSE have been discussed in Ref.B0 inhomogeneities, off-resonance effects, etc. Linear background phase errors were corrected further using information from an external, static agarose reference, which provided high signal-to-noise ratio due to short T1 times . It cannot be excluded that this difference is caused by the vertical orientation of the mouse in the magnet used in Ref.The main aim of this article was to provide detailed global and regional left-ventricular motion patterns. While twist and torsion angles have been reported before for mouse hearts, this study provides for the first time a detailed motion analysis of the entire cardiac cycle in normal, adult C57Bl/6 mice under isoflurane anesthesia using (phase-contrast) MRI, and followed clinical convention for both the different velocity components and the segmental analysis , 26. WhiTwist angles and torsion were similar to the values reported by Henson et al.  and, morThe proof-of-principle application to the ischemia–reperfusion mouse model  demonstrIn conclusion, this work presents an optimized method based on phase-contrast MRI that allows for a comprehensive assessment of regional cardiac function in mouse hearts at 9.4 T. Black-blood contrast was found to be essential to obtain accurate and reproducible velocity encoding. A detailed analysis was performed to establish baseline values of normal transmural wall motion pattern. This technique will help to investigate systolic and diastolic functions in genetically and surgically manipulated mouse models of human heart disease.


InAs/GaSb type II superlattices were grown on (100) GaSb substrates by metalorganic chemical vapor deposition (MOCVD). A plane of mixed As and Sb atoms connecting the InAs and GaSb layers was introduced to compensate the tensile strain created by the InAs layer in the SL. Characterizations of the samples by atomic force microscopy and high-resolution X-ray diffraction demonstrate flat surface morphology and good crystalline quality. The lattice mismatch of approximately 0.18% between the SL and GaSb substrate is small compared to the MOCVD-grown supperlattice samples reported to date in the literature. Considerable optical absorption in 2- to 8-μm infrared region has been realized.PACS: 78.67.Pt; 81.15.Gh; 63.22.Np; 81.05.Ea InAs/GaSb superlattices (SLs) are important for long-wavelength infrared (IR) applications because of their broken gap type II band alignment with the conduction band minimum of InAs lying below the valence band maximum of GaSb. Such type II SL material has been investigated widely as a viable alternative to conventional HgCdTe IR detecting materials due to the unique capability for band structure engineering which results in great flexibility in controlling the detection wavelength from 3 to 30 μm) , low Aug to 30 μmxSbx 1-mixing plane that connects the InAs and GaSb layers as the interfacial layers.One of the key issues during the growth of InAs/GaSb SLs is to introduce suitable interfacial layers between InAs and GaSb to offset their lattice mismatch of 0.59%, since misfit dislocations will be generated as the number of layers in the SL increases. The widely investigated MBE growth of such SLs usually introduces InSb-like interface (IF) to make the SL match the GaSb substrate as much as possible ,12,16. Hin situ sensor. The chamber pressure during growth was 100 Torr. Epi-pure™  trimethylindium and triethylgallium were used as column III precursors and trimethylantimony (TMSb) and arsine (AsH3) were used as column V precursors. Prior to the growth, the substrates were cleaned in HCl to remove native surface oxide and then rinsed in isopropyl alcohol followed by N2 blow-drying. In a typical growth, 100-nm GaSb buffer was firstly deposited at 580°C, then the temperature was ramped down to 520°C for the growth of InAs/GaSb SL. Here, we grew 100-period SLs with nominal structures of 4.5-nm InAs and 3-nm GaSb with a plane of mixed As and Sb atoms connecting the GaSb and InAs as the IF layer. The growth of the IFs was controlled by atomic layer epitaxy-like switching sequence [x value of AsxSbx 1-is 0.18. To obtain the AsSb mixing planes, a 3-s growth interruption with AsH3 flowing was introduced after the growth of InAs layer, then the AsH3 and TMSb switching valves were simultaneously closed and opened, respectively. The TMSb switching valve was left open for 10 s to form the nominal As0.18Sb0.82 plane.InAs/GaSb SLs were grown on (100) GaSb substrates in an Aixtron  MOCVD reactor system equipped with a close-coupled showerhead growth chamber and an EpiTT optical 1 radiation as the source. Polarized Raman scattering measurements were performed in the backscattering geometry at room temperature with a Jobin Yvon  HR800 confocal micro-Raman spectrometer. Three scattering configurations, X, Y' and Z' are defined along the The growth was resumed with the deposition of GaSb. The IF, thus, formed is a GaSb-on-InAs IF. For an InAs-on-GaSb IF, the GaSb surface was firstly smoothed by TMSb flowing for 0.5 s, then As was introduced for 5 s to exchange desired amount of Sb atoms on the GaSb surface followed by InAs deposition. The growth rate was 0.7 Å/s for InAs and 1 Å/s for GaSb layers, respectively. The surface morphologies of InAs/GaSb SLs were characterized by atomic force microscopy  operating in air at room temperature. High-resolution X-ray diffraction  was carried out with Cu KαIn situ reflectance can give us useful information of the growth surface during the epitaxy. Figure in situ reflectance at 633 and 951 nm of a 100-period InAs/GaSb SL sample. Fabry-Perot (FP) oscillations are visible in both reflectance curves during the SL growth. The oscillation amplitude of the 951-nm reflectance damps gradually with the growth time due to the gradually increased absorption of the SL material. Using a VI mode [ VI mode , the per2 scan area. Misfit dislocations due to strain accumulation with the thickness of the grown layer did not generate in our 100-period SL samples. Therefore, according to the in situ monitoring reflectance and the AFM image, the planes of mixed As and Sb atoms that we introduced between InAs and GaSb have offset at least partly the strain arising from the lattice mismatch of 0.6% between InAs and GaSb as we expected.The smooth surface of the sample is also confirmed by the AFM image shown in Figure x value of AsxSbx 1-as the fitting parameter. Figure x = 0.1 and x = 0.18, respectively. It is seen that the zeroth satellite peak overlaps completely with the GaSb (004) peak, i.e., achieving complete strain compensation when x is set to 0.18. Here, the experiment XRD pattern can be fitted well by x = 0.1, close to the optimal 0.18, indicating nearly complete strain compensation in our SL samples.The crystalline properties of the samples were characterized by HRXRD, and a typical pattern around the (004) reflection of GaSb substrate is shown in Figure -1, arising from the quasi-confined longitudinal-optical (LO) phonon modes of GaSb and InAs. Because of the overlap in the frequencies of the LO modes of GaSb and InAs, we are unable to distinguish the LO modes of these two materials. In addition, there are two weak peaks in both -1. The 248-cm-1 peak is assigned to the strongly localized Ga-As longitudinal IF vibration, and the very weak 188-cm-1 peak is assigned to the weakly localized In-Sb longitudinal IF vibration. According to the calculation with a random-element isodisplacement model by Shanabrook et al. [-1 in Figure 0.6Sb0.4 plane. This result is different from our dynamical simulation where the As composition is 0.1. This difference is reasonable due to the inherent simplicity of the model as mentioned by the authors. In the -1 is still visible which is assigned to the transverse optical vibrations confined in the InAs [The interfaces in InAs/GaSb superlattices are different from that in usual III-V SLs such as GaAs/AlGaAs. The change of both group III and group V atoms across the IFs leads to the appearance of GaAs-like or InSb-like bonds at the interface, which do not present in both InAs and GaSb ,24. Becak et al. , the vibthe InAs . The actOur 100-period SL samples show considerable IR absorption in the wavelength range of 2 to8 μm as shown in Figure 0.1Sb0.9 plane connecting InAs and GaSb was introduced as the IF layer to compensate the strain of the SL to the GaSb substrate. The validity of the strain compensation was confirmed by the results of the in situ reflectance, AFM, HRXRD and Raman scattering measurements. The RMS roughness of the surface is 0.7 nm, and the lattice mismatch between the SL and the substrate is 0.18%. Absorption coefficient of approximately 2,000 cm-1 is realized in mid-IR region.We have achieved 100-period InAs/GaSb SLs on (100) GaSb substrates by MOCVD. An As3: arsine; C1: first electron sub-band; FP: Fabry-Perot; FTIR: Fourier-transform infrared; HH1: first heavy hole sub-band; HRXRD: high-resolution X-ray diffraction; IR: infrared; LH1: first electron sub-band; LO: longitudinal-optical; MBE: molecular beam epitaxy; MOCVD: metalorganic chemical vapor deposition; QWIP: quantum well IR photodetectors; RMS: root mean square; SL: superlattice; TMSb: trimethylantimony.AFM: atomic force microscopy; AsHThe authors declare that they have no competing interests.LGL carried out the experimental analysis and drafted the manuscript. SML carried out the experimental design. SL carried out the growth and optimization of InAs/GaSb superlattice. TY and LJW participated in the experimental analysis. FQL and XLY participated in the experimental measurement. BX and ZGW participated in the experimental design. All authors read and approved the final manuscript.